The development of new and more effective chemotherapeutic agents for cancer treatment requires consideration of a variety of factors including cytotoxicity, tumor cell proliferation, invasion and metastasis. Conventional anticancer agents have typically been identified on the basis of their cytotoxicity alone, as the signaling pathways required for the maintenance and driving of the malignant process were not known. These pathways are now being elucidated in terms of signal transduction.
Signal transduction is the processing of chemical signals from the cellular environment through the cell membrane, and may occur through at least three distinct mechanisms: phosphorylation, activation of ion channels, and effector enzyme activation via guanine nucleotide binding protein intermediates.
Linkage of selected signal transduction pathways to malignant behavior has been demonstrated using molecular biologic techniques. Three different guanine nucleotide-binding protein-linked receptors have been transfected into normal recipient cells and upon specific ligand activation caused malignant behavior.
Tumor formation and ligand-specific focus formation was found when the serotonin 1c receptor was placed into NIH-3T3 cells and stimulated with a receptor specific ligand. Julius, et al., Science, 244:1057-1062 (1989). Serotonin stimulation of the transfected 5HT1c receptors resulted in mobilization of calcium, leading to a proposed link between receptor activation of the signal pathway and the biologic function of tumor formation.
A similar outcome was seen when an adrenergic alpha-1B receptor was put into NIH-3T3 and Rat-1 cells. Allen, et al., Proc. Natl. Acad. Sci. USA 88:11354-11358 (1991). In that experiment, catecholamine stimulation of the transfected receptor produced foci formation and increased cellular proliferation in culture as well as tumorigenic behavior in nude mice. Allen, et al. further demonstrated the functional coupling of the transfected receptor to production of total inositol phosphates which can secondarily cause internal release and influx of calcium, suggesting a link between these signal transduction pathways and the malignant response.
Lastly, transfection of the odd-numbered muscarinic receptors into NIH-3T3 cells has been shown to stimulate internal calcium release and uptake, arachidonic acid release, and generation of inositol phosphates. See, Bonner, et al., Neuron, 1:403-410 (1988), Gutkind, et al., Proc. Natl. Acad. Sci. USA, 88:4703-4703 (1991). Conklin, et al., Proc. Natl. Acad. Sci. USA, 85:8698-8702 (1988), and Felder, et al., J. Pharmachol. Exp. Ther., 255:1140-1147 (1990). Additionally, ligand activation of the m1, m3, and m5 subtype muscarinic receptors resulted in tumorigenic foci formation in vitro.
Association of the products of the arachidonic acid cascade and generation or inhibition of malignancy has also been documented. Prostaglandins have been implicated in the initiation and promotion of malignancy. Honn, et al., Prostaglandin 21:833-864 (1981). In these experiments, phorbol esters stimulated the production of PGE.sub.2 and PGF.sub.2a which could be selectively inhibited by treatment with the cyclooxygenase inhibitor, indomethacin. Pharmacologic manipulation of prostaglandin synthesis in animal models led to the inhibition of cancer progression and was the impetus for human trials.
Pharmaceutical inhibition directed to specific pathways, such as arachidonic acid release or calcium influx, offers a new approach to cancer treatment and potentially to cancer prevention. Compound 1 was originally designed as a coccidiostat (U.S. Pat. No. 4,590,201) and later developed as a cancer treatment agent of particular use in the treatment of peritoneal carcinomatosis of ovarian cancer (U.S. Pat. No. 5,132,315, and Kohn, et al., J. Natl. Cancer Inst., 82:54-60 (1990)). Recent studies have shown Compound 1 to be a novel inhibitor of selected signal transduction pathways including those which involve calcium influx, the release of arachidonic acid and the generation of inositol phosphates. See. Kohn, et al. Cancer Res., 52:3208-3212 (1992) and Felder, et al., J. Pharmacol. Exp. Ther., 257:967-971 (1991). ##STR1##
Compound 2 is another agent with selectivity for receptor-mediated calcium entry (RMCE). This compound inhibits RMCE in platelets, endothelial cells and neutrophils, and blocks voltage-gated L-type calcium channels in vascular smooth muscle cells under patch clamp. Merritt, et al., Biochem. J. 271:515-522 (1990). Similarly, compound 1 has been shown to inhibit L- and T-type voltage gated calcium channels. Hupe, et al., J. Biol. Chem., 266:10136-10142 (1991). However, neither study evaluated the effect of their respective agents on malignant cells.
Signaling events are so primary in cellular function that any agent which interferes with signal-effector coupling should be uniformly toxic to normal cells and tissues as well as to malignant and metastatic tissues and cells. However, when nude mice received oral administration of compound 1, no toxicity to normal tissues including connective tissue, mucosal surfaces, and bone marrow was observed. See, Hupe, et al. J. Cell. Physiol., 144:457-466 (1990) and Kohn, et al., Cancer Research, 52:3208-3212 (1992). This suggests that malignant cells have a higher state of dependence upon certain second messenger pathways, rendering them selectively sensitive to compound 1 and related agents.